Methylation detection

Base-specific cleavage and MALDI-TOF MS has recently also been implemented as an effective means for high-throughput quantitative analysis of DNA methyl-ation. The analysis of covalent modifications of nucleotides in genomic DNA, e.g., cytosine methylation, has gained significant momentum when it became apparent that genetic information is not only stored in the arrangement of four nucleotide bases, but also in the covalent modification of selected bases. As indicated above, methylation of cytosine is the most common modification in mammals. The covalent addition of methyl groups to cytosine is catalyzed by the DNA methyltransferase (DNMT) enzyme family (Pradhan et al., 1999; Szyf and Detich, 2001). More specifically, DNA methyltransferases target cyto-sines in CpG dinucleotides. Such CpG dinucleotides are generally underrepre-sented in the human genome and are concentrated in distinct areas called CpG islands. A large proportion of these CpG islands are found in the promoter regions of genes. The conversion of cytosine into 5-methylcytosine in promoter-associated CpG islands causes changes in chromatin structure, usually resulting in transcriptional silencing of the genes controlled by this promoter region.

While the study of the role of methylation changes in the context of cell biology is a rapidly expanding research field, the importance of methylation is already highlighted through its connection to mammalian development, imprinting, X-chromosome inactivation (Li, 2002), suppression of parasitic DNA (Walsh et al., 1998) and cancer etiology (Costello et al, 2000; Costello and Plass, 2001; Feinberg, 2001; Jones and Baylin, 2002). DNA methylation analysis has received particular attention in cancer research, because several studies have shown that changes in the methylation status of nucleosomal DNA promise to be a powerful marker for the early detection of neoplastic events (Tsou et al., 2002; Etzioni et al, 2003; Laird, 2003).

Several methods have been developed to analyze DNA methylation. Because common techniques for amplification of DNA cannot preserve the cytosine methylation profile (the presence of methylated cytosine cannot be mirrored in the PCR product), most methods rely on bisulfate treatment of genomic DNA prior to amplification (Clark et al., 1994). Bisulfite treatment converts all non-methylated cytosine into uracil, whereas all methylated cytosine are inert. This treatment translates the methylation mark into a sequence change. Amplification products of the target region therefore carry the methylation pattern as a pattern of C/T sequence changes. Correspondingly, sequence analysis of PCR products from bisulfate treatment can be used to conclude on the methylation status of CpGs in a genomic region.

We have shown in earlier sections how the mass signal changes of a base-specific cleavage patterns can be used to identify sequence changes. The process of base-specific cleavage lends itself also for the analysis of DNA methylation. The mass signal pattern is affected by methylation through the C/T sequence changes introduced into the bisulfate-treated genomic DNA. The exact change depends on the type of cleavage reaction: methylation can introduce new cleavage sites resulting in new, shorter products (and hence new mass signals with lower mass); methylation can lead to a replacement of an existing cleavage site with a non-cleavable nucleotide and therefore connect two existing fragments together resulting in a new, longer product (new mass signals with higher mass); and methylation can generate a sequence change in an existing cleavage product that does not affect cleavage, but generates a mass shift. To illustrate this, consider bisulfate treatment of a fully unmethylated genomic region.

All cytosines will be converted into uracils, and correspondingly there will no existing cleavage sites in a C-specific cleavage of the forward strand. Figure 2 illustrates the cleavage reactions. However, each methylated cytosine remains a cytosine and it will therefore generate a cleavage site and corresponding cleavage products/mass signals, which allow discovery of the methylation and its identification. Cleavage reactions, in which a methylation event generates a new cleavage site, are perfectly suited for sensitive discovery of methylation.

The third option, in which methylation does not affect a cleavage site but leads to mass shifts, is the most interesting when quantitation of the relative amount of methylated DNA is desired. The T-reverse cleavage, for example, always cleaves outside of a CpG dinucleotide. In this reaction methylation events will be represented as mass shifts of 16 Da (the mass difference between G/A, which represents the reverse strand complement of the C/T changes). If multiple CpG sites are enclosed in one cleavage product, the mass will shift in multiples of 16 Da. The coexistence of mass signals representing methylated DNA and unmethylated DNA in close proximity (in terms of mass difference) enables the estimation of the relative amount of methylated DNA by the use of the peak area ratios.

To experimentally verify the concept, base-specific cleavage and MALDI-TOF MS have been used to successfully reconstruct the methylation pattern of IGF2/H19, a well-described genomic region commonly hemi-methylated (Ehrich et al., 2005b). It has then been used successfully for large-scale quantitative profiling of methylation patterns in lung cancer. In this study, the me-thylation status of 47 promoter regions (1426 CpG sites in total) was assessed quantitatively in 48 lung cancer tissue samples and compared to their normal adjacent lung tissue to identify differentially methylated CpGs that allow accurate classification of samples (Ehrich et al, 2005b). Using the technology described here, this study was completed within a single day (measurement and analysis time).

What are the advantages of the described approach over other technologies? Conventionally, DNA methylation is either analyzed by PCR sequencing or by assays such as methylation-specific PCR (MSP), which target individual CpGs (Herman et al., 1996). PCR sequencing delivers very limited quantitative information and usually has to be supplemented by sequencing from clones to verify the results. To minimize statistical effects, a large number of clones need to be sequenced. This makes the approach very cumbersome and expensive. Assays targeting individual CpGs can provide quantitative information with sufficient precision, but are limited in that they only provide information on the targeted CpG. To obtain a reasonable overview of the methylation status of a genomic region, multitudes of assays have to be developed and optimized, which again is a cumbersome task. Base-specific cleavage and MALDI-TOF MS allow the study of DNA methylation in target regions between 200 and 600 bp in length. A single cleavage reaction can generate quantitative methylation data for up to 70 CpGs. Combined with the high-throughput of MALDI-TOF MS acquisition and analysis, this allows efficient screening of genomic regions for differentially methylated CpGs in large numbers of samples.

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